The goal of tissue engineering is to repair or replace tissues and organs with artificial tissue constructs. Scaffolds are typically used for this process, giving mechanical support, and assisting in cell migration and attachment, cell retention and delivery at the site of repair. The scaffold thus mimics the natural matrix found in the body. In other situations, films or layers of tissue itself may be suitable for the repair or transplant. The cells in such films should thus be as similar as possible to those produced naturally by the body. It is thus important that cells grown into these tissue layers or films are properly aligned, and metabolise to produce factors that the tissue they are destined to replace would produce.
The extracellular matrix is a complex variety of glycoproteins and proteoglycans, which provides tissue integrity, acts as a native scaffold for cell attachment and interaction and acts as a reservoir for growth factors. For most connective tissues, collagen makes up the bulk of the extracellular matrix, where it functions as a structural protein as well as a binding partner for glycans that store growth factors.
Collagen is a family of extracellular matrix proteins, the most abundant being type I found in skin, tendon, bone, corneal, type IV found in all basement membranes and type VII found in the basement membrane of skin, oral mucosa and cornea. These collagen assemblies differ depending on the tissue location and function.
The deposition of a collagen matrix depends on the conversion of denovo synthesised pro-collagen to collagen in the extracellular space immediately before its release into the space. This limiting step for collagen matrix deposition is very slow in vitro, both in monolayer cultures and in three dimensional scaffolds.
For a number of tissue engineering applications, tissue grafts (autografts, allografts or xenografts) are considered to be the ‘gold standards’. However, the limited supply of autografts and certain donor site morbidity restricts their utilisation. The use of allografts and xenografts has also been questioned due to poor success rates, possibilities of immune rejection and potential transmission of disease. To this end, tissue engineering was pioneered as the only viable alternative to the transplantation crisis. Several degradable and non-degradable synthetic materials have been evaluated over the years [2-10]. However, non-degradable synthetic materials may become harmful due to mechanical impingement or infection and require a second operation, whilst the degradation products of biodegradable synthetic materials could be deleterious to the surrounding cells and tissues. Natural biomaterials such as collagen [15-18], gelatin [19, 20] and fibrin [21] have been used as raw materials for scaffold fabrication with promising early results. In particular, the use of collagen as a raw material for scaffold fabrication has been advocated because, as a natural occurring biopolymer that constitutes approximately one third of the total body proteins, it is perceived by the body as a normal constituent rather than foreign matter. Despite advancements in purification methods and analytical assays that have assured low immunogenicity and antigenicity, collagen remains an animal derived by-product and its use in clinical applications can be limited due to concerns of inter-species transmission of disease, especially for collagen extracted from bovine tissues (e.g. Bovine Spongiform Encephalopathy and Creutzfeldt Jakob Disease). In fact, 2-3% of patients have an immune response to collagen implants using collagen derived from land-based animals. For this reason, human recombinant collagen has been investigated for scaffold fabrication. Although several expression systems have successfully produced human recombinant procollagens, in all cases procollagen expression levels have been low (15 mg/ml for mammalian cell culture; 15 mg/ml in yeast; and 60 mg/ml in baculovirus) which was prohibited commercialisation and clinical applications. Moreover, whilst recombinant collagens can be expressed in a thermo-stable triple helical form, they lack specific domains otherwise present in native fibrillar collagens, which can compromise their biological function and further reduce their use. In light of this, it has been predicted that companies developing new implantable products are more likely to focus on human collagen products, rather than on products utilising animal-sourced collagen.
Indeed, advancements in molecular and cell biology have allowed the use of cell-based therapies for tissue engineering and regenerative medicine applications. The concept is that replacement, repair and restoration of function can be accomplished best using cells that will create their own host-specific extracellular matrix. Indeed, cells are professional matrix makers and assemble into large aggregates together with ligands, growth factors and other matrix components with a precision and stoichiometric efficiency that is still unmatched by man-made devices, recombinant technology-derived components or chemical compounds. Cell-based injectable systems and cell-sheets derived from autologous primary cell isolates; from established cell lines; and from a variety of stem cells have been used for numerous clinical targets, including cornea, skin, blood vessel, cartilage, lung, cardiac patch, oesophagus and periodontal applications.
Cultured cells deposit extracellular matrix (ECM) molecules and form cell-to-cell junctions. However, typical proteolytic harvest (by trypsin) digests both deposited ECM and cell-to-cell junctions. In contrast, culture dishes covered with a temperature-responsive polymer allow harvesting of intact cell sheets along with their deposited ECM, by simple temperature reduction. Despite the success of cell sheet tissue engineering in regenerative medicine, this technology has still not taken off primarily due to the substantial long period of time required to culture the cells and develop an implantable cell-sheet.
Herein, for first time, we describe the production of cell-sheets within 24-48 h from human cells using a biophysical approach that governs the intra- and extra-cellular milieu in multicellular organisms, termed macromolecular crowding, that invites cells to create their own matrices. The principal that the approach is based on is that the deposition of a collagen matrix depends on the conversion of de novo synthesised procollagen to collagen in the crowded extracellular space or immediately before its release into the same. The rate limiting step of collagen I deposition is the proteolytic conversion of procollagen to collagen. This step is catalysed by procollagen C-proteinase and proteolytic modification of its allosteric regulator. In vivo, the extracellular space is highly crowded (FIG. 1); even dilute body fluids are highly crowded: blood contains 80 g/l protein; urine contains 36-50 g/l solids, and the conversion of procollagen to collagen takes place very fast (FIG. 2). However, in vitro cells are grown in highly dilute conditions; this, in the human body, would represent a medical pathology. However, this situation can be remedied, by adding macromolecules of defined hydrodynamic radius to culture media and thus creating excluded volume effects with defined volume fraction occupancies. Conventional cell culture systems are far from crowded environments (Table 1) and in this dilute, far from physiological, environment the deposition of matrix is very tardy.
TABLE 1Concentration of solids in conventional cell culture systemSOLIDMEDIACOMPANYCONCENTRATIONMinimumInvitrogen, Cat. No: 10370021,11.52 g/lEssential10370039, 10370047, 10370054, Medium10370070F12 NutrientInvitrogen, Cat. No: 2176502911.87 g/lMixtureRPMI 1640Invitrogen, Cat. No. 11835030,12.39 g/lMedium11835055, 11835063, 11835071Ham F10Invitrogen, Cat. No. 22390017,16.55 g/lNutrient Mixture22390025DMEM: F12ATCC, Cat. No. 30-200616.78 g/lMediumDMEM HighInvitrogen, Cat. No. 41965039, 17.22 g/lGlucose (4.5 g/)41965047and L-glutamine
In an attempt to modulate the in vitro micro-environment and closely emulate the in vivo setup, macromolecules have been used to crowd the culture media (Table 2). Under crowded conditions, thermodynamic activities increase by several orders of magnitude and biological processes, such as enzymatic activities and protein folding can be dramatically accelerated.
TABLE 2Macromolecules that have been used to-date as crowding agentsCROWDER USEDRESULTSREFERENCESucrose and glucoseIneffectiveZimmermann and(monomers forMMC significantlyHarrison, 1987, dextran andFicoll ™), PEG 0.2 Kdaincreased enzymaticPNAS, 84: 1871-Ficoll ™ 70 Kda,activity1875DextranT70 Kda, PEG 8 Kda,PEG 35 KDaFicoll ™ 70 KDa,Faster protein foldingvan den Berg et al., Dextran 70 KDarates1999, EMBO Journal, 18(24): 6927-6933PEG 3.5 Kda, Ficoll ™MMC dramaticallyMunishkina et al., 70 KDaincrease fibrillation of2008 Biochemistry, unfolded proteins47(34): 8993-9006Ficoll ™ 70 Kda,MMC dramaticallyZhou et al., 2009, Dextran 70 KDaaccelerated the Journal of nucleation step of fibril Biological Chemistry,formation of human 284(44): Tau fragment & prion 30148-30158proteinDextran Sulphate 500 KdaDramatically Lareu et al., 2007 (negative; 46 nm radius)acceleratedFEBS letters, 581: PSS 200 Kdaextracellular matrix2709-2714(negative: 22 nm radius)compositionLareu et al., 2007 Ficoll ™ 400 KdaBBRC, 363: 171-177(neutral; 4.5 nm radius)Lareu et al., 2007 Ficoll ™ 70 KdaTissue Engineering.,(neutral; 3 nm radius)13(2): 385-391
Animal extracted and recombinant collagen is used in several tissue engineering applications since under appropriate conditions of temperature; pH; and ionic strength since they will self-assemble to produce collagen fibres indistinguishable from fibres found in vivo. However, animal extracted collagens are responsible for immune response, whilst recombinant collagen is not biologically active since it is not produced with the appropriate post-translation modifications.
Lareu et at (2007) describe a dramatic enhancement in collagen matrix deposition by using large negatively charged polameric macromolecules to create an excluded volume effect in long fibroblast cultures. Chen et at (2004) similar results were achieved using a cocktail of macromolecules using dextran sulphate and FICOLL™ (neutral branched hydrophilic polysaccharides) molecules. Again the crowders are negatively charged molecules with large hydrodynamic radii. Similarities have been shown with neutral and negatively charged dextran sulphate by Lareu (2007) and Peng and Raghunath.
[In Press]
The present inventors have surprisingly found that by using poly-dispersed macromolecular crowders, that cell metabolism and extracellular matrix production can be enhanced to such a level that significant quantities of tissue substitute films are produced after as little as 48 hours. It has never previously been shown that poly-dispersity of a macromolecule is key to enhancing the production of tissue substitutes. This knowledge allows the identification of molecules which are suitable for this purpose and the mixing of molecules to enhance the polydispersity of a mixture of molecules.